HUMAN DNA TOPOISOMERASE I-ALPHA

Abstract: Disclosed is a human hTopI- alpha polypeptide and DNA (RNA) encoding such hTopI- alpha polypeptide. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies and antagonists/inhibitors against such polypeptide. Also provided are methods of using the antibodies and antagonist inhibitors to inhibit the action of hTopI- alpha for therapeutic purposes such as an antitumor agent, to detect an autoimmune disease, or retroviral infections and to treat adenocarcinoma of the colon.

Description: HUMAN DNA TOPOISOMERASE I - ALPHA This invention relates to newly identified polynucleotides, polypeptideε encoded by such polynucleotideε, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptideε. More particularly, the polypeptide of the present invention iε human DNA topoisomerase I alpha (hTopI-α). The invention also relates to inhibiting the action of such polypeptides. DNA topoisomerase I and II catalyze the breaking and rejoining of DNA strands in a way that allows the strands to pass through one another, thus altering the topology of DNA. Type I topoisomerase recognizes double-stranded DNA but only breaks on strand in the process of relaxing DNA, while the type II enzyme breaks both strands of duplex DNA. Both enzymes can perform a variety of similar topological inter- conversions, including relaxation of super coiled DNA, knotting and unknotting and catenation and decatenation of duplex DNA. Topoisomerase I is ATP-independent, while Topoisomerase II requires energy. Both topoisomerase I and II can provide the topological inter-conversions necessary for transcription and replication. For example, topoisomerase I can provide the necessary unlinking activity for efficient in vitro DNA -1- replication (Minden, et al., J. Biol. Chem., 260:9316, (1985)), however, topoisomerase II can also facilitate the .replication of SV40 DNA by HeLa cell lysates (Yang, et al.. Proceedings of the National . Academy of Sciences, U.S.A., 84:950, (1987)). Genetic studies in yeast reveal that both replication and transcription proceed in single mutants deficient in either topoisomerase I or II (Goto, et al.. Proceedings of the National Academy of Sciences, U.S.A., 82:7178 (1985)). In cells lacking both topoisomeraseε, transcription and replication are dramatically reduced (Uemura, et al., EMBO Journal, 5:1003 (1986)). Several lines of evidence suggest that topoisomerase I normally functions during transcription. The enzyme has been shown to be localized preferentially to actively transcribed loci by im unofluorescence (Fleish ann, et al. , Proceedings of the National Academy of Sciences, U.S.A., 81:6958 (1984)), and by co-immunoprecipitation with transcribed DNA (Gilmore, et al., Cell, 44:401, (1986)). Furthermore, topoisomerase I cleavage sites have been mapped to regions in and around transcribed DNA (Bonner, et al.. Cell, 41:541 (1985)). Nonetheless, at least in yeast, topoisomerase II can apparently substitute for the functions of topoisomerase I in transcription. While all cells utilize Topoisomerase I and II for transcription and replication, cells with a high amount of transcription and replication, eg. cancerous cells, have a much higher concentration of Topoisomerase I and II. Topoisomerase I has been used to classify autoimmune disease. Autoimmune diseases are diseases in which an animal's immune system attacks its own tissues. Often the various types of autoimmune disease can be characterized based upon the specificity of autoantibodies which are produced. For example, it is well known that the serum of patients having the connective tissue autoimmune disease progressive systemic sclerosis, also known aε scleroderma, frequently contain antibodies to such nuclear antigens as topoisomerase I. Thus, the ability to accurately detect the presence of antibodieε reactive with topoisomerase I can greatly assist in evaluating the prognosis and planning, or monitoring, of the appropriate therapy for patients with scleroderma. A 3645-base pair human topoisomerase I cDNA clone and a mutated version of the cDNA encoding a protein with phenylalanine instead of tyrosine at position 723 have been overexpressed two to five fold in stably transfected baby hamster kidney cells. The results of this overexpression indicate that tyrosine 723 is essential for enzyme activity and is consistent with predictions based on homology comparisons with the yeast enzymes, that this is the active- site tyrosine in the human topoisomerase I. (Madden, K.R. and Champoux, J.J., Cancer Research, 52:525-532, (1992)). Also, cDNA clones encoding human topoisomerase I have been isolated from an expression vector library screened with autoimmune anti-topoisomerase I serum. The sequence data shows that the catalytically active 67.7-kDa fragment iε comprised of the carboxyl terminus, (D'Arpa, P. et al., Proc. Natl. Acad. Sci. U.S.A., 85:2543-2547, (1988)). cDNA molecules coding for eukaryotic topoisomerase I polypeptide which encode at least one epitope for autoantibodies to eukaryotic topoisomerase I and cloning vehicles capable of expressing these cDNA molecules are disclosed in United States Patent No. 5,070,192. In accordance with one aspect of the present invention, there are provided novel mature polypeptides which are hTopI- α, as well as fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin. In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides. In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques . In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides, which may be used diagnostically to detect cancers and autoimmune diseases. In accordance with yet another aspect of the present invention, there are provided antagonist/inhibitors to such polypeptides, which may be used therapeutically to inhibit the action of such polypeptides, for example, aε an antitumor agent and to detect autoimmune diεeases, and to treat adenocarcinoma of the colon, and retroviral infections. These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein. The following drawingε are illuεtrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims. Fig. 1 shows the cDNA and corresponding deduced amino acid sequence of hTopI-α. The polypeptide encoded by the amino acid sequence shown is the mature form of the polypeptide. The standard one letter abbreviation for amino acids iε uεed. Fig. 2 shows a comparison of hTOPI-α and human topoisomerase I at the amino acid level. The upper line is hTOPI-α. In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75714 on March 18, 1994. A polynucleotide encoding a polypeptide of the present invention was obtained from a fetal brain cDNA library. It is homologous to human topoiεomerase I. It contains an open reading frame encoding a protein of approximately 601 amino acid residues and it is structurally related to human DNA topoisomerase I showing 86% similarity and 70% identity at the amino acid level. Further, hTopI-α shows 83% similarity and 67% identity to human topoisomerase I as published by D'Arpa et al. The amino acid tyrosine 723 . The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1 or the deposited cDNA. The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such aε introns or non-coding sequence 5 ' and/or 3 ' of the coding sequence for the mature polypeptide. Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide. Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotideε which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variantε include deletion variantε, subεtitution variantε and addition or inεertion variantε. As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence εhown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant iε an alternate form of a polynucleotide sequence which may have a εubεtitution, deletion or addition of one or more nucleotideε, which does not substantially alter the function of the encoded polypeptide. The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the ' influenza hemagglutinin protein (Wilson, I., et al.. Cell, 37:767 (1984)). The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotideε in a preferred embodiment encode polypeptideε which retain εubstantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA. The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the depoεited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any deεcription of sequenceε herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted. The present invention further relates to a hTopI-α polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide. The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. The polypeptide of the preεent invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such aε a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which iε employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural syεtem, iε isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptideε could be part of a composition, and still be iεolated in that εuch vector or composition is not part of its natural environment. The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cellε can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the hTopI-α genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host. The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such -9- SUBSTTTUTE SHEET (RULE 26) procedures and others are deemed to be within the scope of those skilled in the art. The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expresεion vector alεo containε a riboεome binding εite for translation initiation and a transcription terminator. The vector may also include appropriate sequenceε for amplifying expreεsion. In addition, the expreεεion vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resiεtance in E. coli. The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to expreεε the protein. As representative examples of appropriate hostε, there may be mentioned: bacterial cells, such aε E. coli. Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cellε such as Drosophila and Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. More particularly, the present invention also includes recombinant constructs comprising one or more of the εequences as broadly described above. The constructε compriεe a vector, εuch aε a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of εuitable vectorε and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plasmid or vector may be used as long as they are replicable and viable in the host. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRε from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)). The conεtructs in hoεt cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoterε. Cell-free translation εystems can also be employed to produce such proteins uεing RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the discloεure of which is hereby incorporated by reference. Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes iε increaεed by inεerting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase itε transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenoviruε enhancers. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin reεistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymeε εuch aε 3-phosphoglycerate kinase (PGK), cc-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences. and preferably, a leader εequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologouε sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus εubtiliε. Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmidε compriεing genetic elementε of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or uεe of cell lysing agents, such methods are well know to those skilled in the art. Various mammalian cell culture systems can also be employed to expresε recombinant protein. Exampleε of mammalian expreεεion systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expresεion vectorε will compriεe an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding siteε, polyadenylation εite, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequenceε derived from the SV40 εplice, and polyadenylation εiteε may be uεed to provide the required nontranscribed genetic elements. The polypeptideε can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. The polypeptides of the preεent invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques -14- SUBSTITUTESHEETtRULE26) from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue. The polypeptides of the present invention are useful for identifying other molecules which have similar biological activity. An example of a screen for this is isolating the coding region of the hTopI-α gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to. This invention also provides a method of screening drugs to identify drugs which specifically interact with, and bind to., hTopI-α which comprises contacting a mammalian cell comprising a DNA molecule encoding hTOPI-α with a plurality of drugs, detecting those drugs which bind to the mammalian cell, and thereby identifying drugs which specifically interact with, and bind to, hTOPI-α. Various methodε of detection may be employed. The drugs may be "labeled" by association with a detectable marker substance (e.g., radiolabel or a non-isotopic label such as biotin) . Drug candidates are identified by choosing chemical compounds which bind with high affinity to the expressed hTOPI-α protein in transfected cells, using radioligand binding methods well known in the art. The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromoεomal location. The mapping of DNAs to chromosomeε according to the preεent invention is an important first step in correlating those sequences with genes associated with disease. Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for asεigning a particular DNA to a particular chromosome. Using the preεent invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragmentε from εpecific chromoεomeε or poolε of large genomic cloneε in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-εorted chromoεomeε and preεelection by hybridization to conεtruct chromosome specific-cDNA libraries. Fluorescence in situ hybridization (FISH) of a cDNA clones to a etaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as εhort aε 500 or 600 bases; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good resultε a reasonable percentage of the time. For a review of this technique, see Ver a et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988). Once a sequence has been mapped to a preciεe chromoεomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johnε Hopkins University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative geneε. (Thiε assumes 1 megabase mapping resolution and one gene per 20 kb). The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expreεεing them can be uεed as an immunogen to produce antibodies thereto. Theεe antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedureε known in the art may be used for the production of such antibodies and fragments. Antibodieε generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptideε. Such antibodieε can then be used to isolate the polypeptide from tissue expressing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodieε produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. The present invention also relates to a diagnostic assay for detecting the concentration of hTOPI-α in a sample from a host. An example of such an asεay is an ELISA assay which utilizes an antibody εpecific to an hTOP-Iα antigen, preferably a monoclonal antibody, which is coupled to an indicator enzyme εuch as horseradish peroxidase to generate a very specific and sensitive asεay syεtem. After binding of the peroxidase-coupled antibody to the antigen, the peroxidase can be used to generate a colored product that is measurable and whose concentration is related to the amount of antigen in a εample. Because of the catalytic nature of the enzyme the syεtem greatly amplifies the εignal. A high level of oxalyl-CoA decarboxylase is indicative of cancer since some human colon carcinoma cells have increased levels of hTopI-α. They may also be indicative of autoimmune diseases, such as εcleroderma, rheumatoid arthritiε and AIDS related complex. Other exampleε of immunoaεsays which can be used to detect the level of antibodies against the polypeptide of the present invention are competitive and non-competitive -18- SUBSTITϋTE SHEET (RULE 26) immunoassays in either a direct or indirect format. Examples include the radioimmunoassay, the sandwich (immunometric) .assay and the Western blot assay. Detection of antibodies which bind to the hTopI-α of the invention can be done utilizing immunoassays which run in either the forward, reverse or simultaneous modes, including immunohistochemical assays on physiological samples. Regardlesε of the type of immunoassay used, the concentration of hTopI-α utilized can be readily determined by one of ordinary skill in the art using routine experimentation. The hTopI-α of the invention can be labeled and bound to many different carriers and used to detect the presence of antibody specifically reactive with the polypeptide. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylaseε, natural and modified celluloεe, polyacrylamides, agarose and magnetite. The nature of the carrier can be either εoluble or insoluble. The present invention iε alεo directed to antagonist/inhibitor molecules of the polypeptides of the present invention which may be used to reduce or eliminate the function of the polypeptide. An example of an antagonist is an antibody, or in some cases, an oligonucleotide which binds to the hTopI-α polypeptides. An example of an inhibitor is a small molecule which binds to and occupies the catalytic site of the polypeptide thereby making the catalytic site inaccessible to substrate such that normal biological activity is prevented. Examples of small molecules include but are not limited to small peptides or peptide-like moleculeε. Another example of an inhibitor iε an antisense construct which inhibits hTopI-α in vivo by the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodeε for the mature polypeptideε of the preεent invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al. Science, 241:456 (1988); and Dervan et al. , Science, 251: 1360 (1991)), thereby preventing transcription and the production of hTOPI-α. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the hTopI-α (antisenεe - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expresεion, CRC Press, Boca Raton, FL (1988)). The antagonist/inhibitorε may be uεed to treat tumorε since specific inhibition of htopl-α will inhibit tumor cell growth by blocking tumor cell DNA replication. The antagonist/inhibitors may also be used to treat retroviral infections by inhibiting hTopI-α and therefore blocking initiation and replication of the virus. The antagonist inhibitors may also be used to treat adenocarcinoma of the colon, εince metaεtaεes are prevented by blocking DNA transcription of the cancerous cellε. The present invention iε alεo directed to an assay using hTopI-α to identify antagonist/inhibitors of hTopl-α and/or human Topoisomerase I. DNA, hTopl-α and a potential antagonist/inhibitor could be combined together under appropriate conditions for a length of time sufficient for hTopl-α to act on the single strand DNA. The DNA could then be analyzed, for example, by gel electrophoresis, to determine whether hTopl-α functioned properly and in this way it could be determined whether there was an effective antagonist/inhibitor. -20- 3UBSTITUTE SHEET (RULE 26) The compounds, e.g., antagonist/inhibitor compounds, of the present invention, may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the antagonist/inhibitor, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptideε of the preεent invention may be employed in conjunction with other therapeutic compoundε. The pharmaceutical compoεitionε may be administered in an effective amount to effectively inhibit hTopl-α from facilitating DNA transcription and replication, in the case where this action leads to undeεired conditions, eg. retroviral infections. The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unlesε otherwise specified, are by weight. In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described. "Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan. "Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment iε uεed with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 °C are ordinarily used, but may vary in accordance with the supplier's instructionε. After digeεtion the reaction iε electrophoreεed directly on a polyacrylamide gel to iεolate the desired fragment. Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980). "Ligation" refers to the proceεε of forming phoεphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al. , Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 μg of approximately equi olar amounts of the DNA fragments to be ligated. Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973). Example 1 Expression of Recombinant hTopl-α in COS cells The expresεion of plaεmid, pcDNAtopI HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire hTopI-αprecursor and a HA tag fused in frame to its 3 ' end waε cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Ni an, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope. The plasmid construction εtrategy iε deεcribed as followε: The expresεion plaεmid pcDNATopI-α ATCC # 75714, encoding for hTopl-α waε constructed by PCR on the pBLTopI-α using two primers: the 5' primer 5' - CGGGATCCATGCGCGTGGTGCGG - 3' contains a bAM hi site followed by 15 nucleotides of HhTopI-α coding sequence starting from the initiation codon; the 3' sequence 5' CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGAATTCAAAGTCTTCTCC - 3' contains complementary sequenceε to an Xba I site, tranεlation stop codon, HA tag and the last 18 nucleotides of the hTopl-α coding sequence (not including the stop codon). Therefore, the PCR product contains a Bam HI site, active hTopl-α coding sequence followed by HA tag fused in frame, a -23- SUBST1TUTESHEET(RULE26) translation termination stop codon next to the HA tag, and an Xba I site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digeεted with Bam HI and Xba I reεtriction enzyme and ligated. The ligation mixture waε tranεformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resiεtant colonies were selected. Plasmid DNA was isolated from transformantε and examined by restriction analysis for the presence of the correct fragment. For expreεεion of the recombinant hTopl-α, COS cells were transfected with the expresεion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). The expression of the hTopl-α HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35 S-cysteine two days post transfection. Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al.. Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels. Example 2 In vitro transcription and tranεlation of hTopl-α The in vitro transcription and translation of the hTopI- α waε carried out uεing the TNT Coupled Reticulocyte Lysate System (Promega, Madison, WI). The plasmid vector used is pBLSK. The cDNA encoding for hTopl-α was cloned directionally EcoRI to Xhol with the EcoRI site defining the 5' end of the gene and the Xhol site defining the 3' end of the gene. The gene was inεerted in the T3 direction. T3 -24- SUBSTlTUTE SHEET (RULE 26) defines a bacteriophage RNA polymerase which recognizeε a specific promoter, and transcribes the DNA into a mRNA. One microgram of the pBLSKhTOPIα was incubated with 25 μl of TNT rabbit reticulocyte lysate, 2 μl TNT reaction buffer, 1 μl T3 RNA polymerase, 1 μl of amino acid mixture minus ethionine (lmM), 4 μl of 35 S-methionine (1,OOOCi/mmol) at lOmCi/ l, 1 μl RNasin ribonuclease inhibitor (40U/μl) in 50 μl of final volume at 37°C for 1.5 hour. 5 μl of the reaction mixture was mixed with loading buffer, boiled for 5 minutes and loaded on a 10% SDS polyacrylamide gel to separate the protein. The gel was then fixed 10% acetic acid, 10% methanol at room temperature for 30 minutes, soaked in Amplify solution (Amersham) at room temperature for 1.5 hours, dried, and subjected to autoradiograph. The obεerved molecular weight of the hTopl-α in thiε εystem is 70 kD, which agrees with the predicted molecular weight by the sequence. Example 3 Expresεion pattern of hTopl-α in human tissue Northern blot analysis was carried out to examine the levels of expression of hTopIα in human tissues. Total cellular RNA samples were isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033). About lOμg of total RNA isolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatiε, Molecular Cloning, Cold Spring Harbor Preεε, (1989)). The labeling reaction was done according to the Stratagene Prime- It kit with 50ng DNA fragment. The labeled DNA was purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filter was then hybridized with radioactive labeled full length hTopl-α gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 β C. After wash twice at room temperature and twice at 60°C with 0.5 x SSC, 0.1% SDS, the filter was then exposed at -70°C overnight with an intenεifying screen. The message RNA for hTopIα is present in all the tissues with abundance in ovary, testes, lung, spleen and prostate. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: WEI, ET AL. (ii) TITLE OF INVENTION: Human Topoisomerase I-α (iϋ) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN (B) STREET: 6 BECKER FARM ROAD (C) CITY: ROSELAND (D) STATE: NEW JERSEY (E) COUNTRY: USA (F) ZIP: 07068 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5 INCH DISKETTE (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: WORD PERFECT 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: SUBMITTED HEREWITH (C) CLASSIFICATION: (vii) ATTORNEY/AGENT INFORMATION: (A) NAME: FERRARO, GREGORY D. (B) REGISTRATION NUMBER: 36,134 (C) REFERENCE/DOCKET NUMBER: 325800-104 (viii) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1,917 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: cDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: GCAGATGCGC GTGGTGCGGC TGCTGCGGCT CCGGGCGGCT CTGACGCTGC TCGGGGAGGT 60 CCCCCGCCGC CCGGCCTCCC GGGGTGTCCC GGGCTCGCGC AGGACGCAGA AGGGCAGTGG 120 AGCCAGGTGG GAGAAGGAGA AGCACGAAGA CGGGGTGAAG TGGAGACAGC TGGAGCACAA 180 GGGCCCGTAC TTCGCACCCC CATACHAGCC CCTTCCCGAC GGAGTGCGTT TCTTCTATGA 240 AGGAAGGCCT GTGAGATTGA GCGTGCCAGC GGAGGAGGTC CCCACTTTTT ATGGGAGGAT 300 GTTAGATCAT GAATACACAA CAAAGGAGGT TTTCCGGAAG AACTTCTTCA ATGACTGGCG 360 AAAGGAAATG GCGGTGGAAG AGAGGGAAGT CATCAAGAGC CTGGACAAGT GTGACTTCAC 420 GGAGATCCAC AGATACTTTG TGGACAAGGC CGCAGCCCGG AAAGTCCTGA GCAGGGAGGA 480 GAAGCAGAAG CTAAAAGAAG AGGCAGAAAA ACTTCAGCAA GAGTTCGGCT ACTGTATTTT 540 AGATGGTCAC CAAGAAAAAA TAGGCAACTT CAAGATTGAG CCGCCTGGCT TGTTCCGTGG 600 CCGTGGCGAC CATCCCAAGA TGGGGATGCT GAAGAGAAGG ATCACGCCAG AGGATGTGGT 660 TATCAACTGC AGCAGGGACT CGAAGATCCC CGAGCCGCCG GCGgGGCACC AGTGGAAGGA 720 GGTGCGCTCC GATAACACCG TCAXGTGGCT GGACAGCTTG ACCGAGAGCG TTCAGAACTC 780 CATCAAGTAC ATCATGCTGA ACCCTTGCTC GAAGCTGAAG GGGGAGACAG CTTGGCAGAA 840 GTTTGAAACA GCTCGACGCC TGCGGGGATT TGTGGACGAG ATCCGCTCCC AGTACCGGGC 900 TGACTGGAAG TCTCGGGAAA TGAAGACGAG ACAGCGGGCG GTGGCCCTGT ATTTCATCGA 960 TAAGCTGGCA CTGAGAGCAG GAAATGAGAA GGAGGACGGT GAGGCGGCCG ACACCGTGGG 1020 CTGCTGTTCC CTCCGCGTGG AGCACGTCCA GCTGCACCCG GAGGCCGATG GTTGCCAACA 1080 CGTGGTGGAA TTTGACTTCC TGGGGAAGGA CTGCATCCGC TACTACAACA GAGTGCCGGT 1140 GGAGAAGCCG GTGTACAAGA ACTTACAGCT CTTTATGGAG AACAAGGACC CCCGGGACGA 1200 CCTCTTCGAC AGGCTGACCA CGACCAGCCT GAACAAGCAC CTCCAGGAGC TGATGGACGG 1260 GCTGSCGGCC AAGGTGTTCC GGACCTACAA CGCCTCCATC ACTCTGCAGG AGCAGCTGCG 1320 GGCCCTGACG CGCGCCGAGG ACAGCATAGC AGCTAAGATC TTATCCTACA ACCGAGCCAA 1380 CCGAGTCGTG GCCATTCTCT GCAACCATCA GCGAGCAACC CCCAGTACGT TCGAGAAGTC 1440 GATGCAGAAT CTCCAGACGA AGATCCAGGC AAAGAAGGAG CAGGTGGCTG AGGCCAGGGC 1500 AGAGCTGAGG AGGGCGAGGG CTGAGCACAA AGCCCAAGGG GATGGCAAGT CCAGGAGTGT 1560 CCTGGAGAAG AAGAGGCGGC TCCTGGAGAA GCTGCAGGAG CAGCTGGCGC AGCTGAGTGT 1620 GCAGGCCACG GACAAGGAGG AGAACAAGCA GGTGGCCCTG GGCACGTCCA AGCTCAACTA 1680 CCTGGACCCC AGGATCAGCA TTGCCTGGTG CAAGCGGTTC AGGGTGCCAG TGGAGAAGAT 1740 CTACAGCAAA ACACAGCGGG AGAGGTTCGC CTGGGCTCTC GCCATGGCAG GAGAAGACTT 1800 TGAATTCTAA CGACGAGCCG TGTTGAAACT TCTTTTGTAT GTGTGTGTGT TTTTTTCACT 1860 ATTAAAGCAG TACTGGGGAA TTTTGTACAA TAAAAAAAAA AAAAAAAAAA AAAAAAA 1917 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 601 AMINO ACIDS (B) TYPE: AMINO ACID (C) STRANDEDNESS: (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PROTEIN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Arg Val Val Arg Leu Leu Arg Leu Arg Ala Ala Leu Thr Leu 5 10 15 Leu Gly Glu Val Pro Arg Arg Pro Ala Ser Arg Gly Val Pro Gly 20 25 30 Ser Arg Arg Thr Gin Lys Gly Ser Gly Ala Arg Trp Glu Lys Glu 35 40 45 Lys Hiε Glu Aεp Gly Val Lyε Trp Arg Gin Leu Gly His Lys Gly 50 55 60 Pro Tyr Pag Ala Pro Pro Tyr Glu Pro Leu Pro Asp Gly Val Arg 65 70 75 Phe Phe Tyr Glu Gly Arg Pro Val Arg Leu Ser Val Pro Ala Glu 80 85 90 Glu Val Pro Thr Phe Tyr Gly Arg Met Leu Asp His Glu Tyr Thr 95 100 105 Thr Lys Glu Val Phe Arg Lys Asn Phe Phe Asn Asp Trp Arg Lys 110 115 120 Glu Met Ala Val Glu Glu Arg Glu Val lie Lys Ser Leu Asp Lys 125 130 135 Cys Asp Phe Thr Glu lie Hiε Arg Tyr Phe Val Asp Lys Ala Ala 140 145 150 Ala Arg Lyε Val Leu Ser Arg Glu Glu Lys Gin Lys Leu Lys Glu 155 160 165 Glu Ala Glu Lys Leu Gin Gin Glu Phe Gly Tyr Cys lie Leu Asp 170 175 180 Gly His Gin Glu Lyε lie Gly Asn Phe Lys lie " Glu Pro Pro Gly 185 190 195 Leu Phe Arg Gly Arg Gly Asp His Pro Lys Met Gly Met Leu Lys 200 205 210 Arg Arg lie Thr Pro Glu Aεp Val Val lie Aεn Cyε Ser Arg Asp 215 220 225 Ser Lyε lie Pro Glu Pro Pro Ala Gly Hiε Gin Trp Lyε Glu Val 230 235 240 Arg Ser Aεp Aεn Thr Val Thr Trp Leu Ala Ala Trp Thr Glu Ser 245 250 255 Val Gin Asn Ser lie Lys Tyr lie Met Leu Aεn Pro Cyε Ser Lys 260 265 270 Leu Lys Gly Glu Thr Ala Trp Gin Lys Phe Glu Thr Ala Arg Arg 275 280 285 Leu Arg Gly Phe Val Asp Glu lie Arg Ser Gin Tyr Arg Ala Asp 290 295 300 Trp Lys Ser Arg Glu Met Lys Thr Arg Gin Arg Ala Val Ala Leu 305 310 315 Tyr Phe lie Asp Lys Leu Ala Leu Arg Ala Gly Asn Glu Lys Glu 320 325 330 Asp Gly Glu Ala Ala Asp Thr Val Gly Cys Cys Ser Leu Arg Val 335 340 345 Glu His Val Gin Leu His Pro Glu Ala Asp Gly Cys Gin His Val 350 355 360 Val Glu Phe Asp Phe Leu Gly Lys Aεp Cys lie Arg Tyr Tyr Asn 365 370 375 Arg Val Pro Val Glu Lys Pro Val Tyr Lys Asn Leu Gin Leu Phe 380 385 390 Met Glu Aεn Lys Asp Pro Arg Asp Asp Leu Phe Asp Arg Leu Thr 395 400 405 Thr Thr Ser Leu Asn Lys His Leu Gin Glu Leu Met Asp Gly Leu 410 415 420 Thr Ala Lys Val Phe Arg Thr Tyr Asn Ala Ser lie Thr Leu Gin 425 430 435 Glu Gin Leu Arg Ala Leu Thr Arg Ala Glu Asp Ser lie Ala Ala 440 445 450 Lys lie Leu Ser Tyr Asn Arg Ala Asn Arg Val Val Ala lie Leu 455 460 465 Cys Asn His Gin Arg Ala Thr Pro Ser Thr Phe Glu Lyε Ser Met 470 475 480 Gin Aln Leu Gin Thr Lyε lie Gin Ala Lyε Lyε Glu Gin Val Ala 485 490 495 Glu Ala Arg Ala Glu Leu Arg Arg Ala Arg Ala Glu Hiε Lys Ala 500 505 510 Gin Gly Asp Gly Lys Ser Arg Ser Val Leu Glu Lys Lys Arg Arg 515 520 525 Leu Leu Glu Lys Leu Gin Glu Gin Leu Ala Gin Leu Ser Val Gin 530 535 540 Ala Thr Asp Lys Glu Glu Asn Lys Gin Val Ala Leu Gly Thr Ser 545 550 555 Lys Leu Asn Tyr Leu Asp Pro Arg lie Ser lie Ala Trp Cys Lys 560 565 570 Arg Phe Arg Val Pro Val Glu Lyε lie Tyr Ser Lyε Thr Gin Arg 575 580 585 Glu Arg Phe Ala Trp Ala Leu Ala Met Ala Gly Glu Aεp Phe Glu 590 595 600 Phe

Claims: WHAT IS CLAIMED IS: 1. An isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding an hTopl-α polypeptide having the deduced amino acid sequence of Figure 1 or a fragment, analog or derivative of said polypeptide; (b) a polynucleotide encoding an hTopl-α polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75714 or a fragment, analog or derivative of said polypeptide. 2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA. 3. The polynucleotide of Claim 1 wherein the polynucleotide iε RNA. . The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA. 5. The polynucleotide of Claim 2 wherein said polynucleotide encodes hTopl-α having the deduced amino acid sequence of Figure 1. 6. The polynucleotide of Claim 2 wherein said polynucleotide encodes an hTopl-α polypeptide encoded by the cDNA of ATCC Deposit No. 75714. 7. The polynucleotide of Claim 1 having the coding sequence of hTopl-α as shown in Figure 1. 8. The polynucleotide of Claim 2 having the coding sequence of hTopl-α depoεited aε ATCC Depoεit No. 75714. 9. A vector containing the DNA of Claim 2. 10. A host cell genetically engineered with the vector of Claim 9. 11. A proceεε for producing a polypeptide comprising: expressing from the host cell of Claim 10 the polypeptide encoded by said DNA. 12. A procesε for producing cellε capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 9. 13. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having hTopl-α activity. 14. A polypeptide selected from the group consisting of (i) a hTopl-α polypeptide having the deduced amino acid sequence of Figure 1 and fragmentε, analogs and derivatives thereof and (ii) a hTopl-α polypeptide encoded by the cDNA of ATCC Deposit No. 75714 and fragments, analogs and derivatives of said polypeptide. 15. The polypeptide of Claim 14 wherein the polypeptide is hTopl-α having the deduced amino acid sequence of Figure 1. 16. An antibody against the polypeptide of claim 14. 17. An antagonist/inhibitor against the polypeptide of claim 14. 18. A method for the treatment of a patient having need to inhibit hTopl-α comprising: adminiεtering to the patient a therapeutically effective amount of the antagoniεt/inhibitor of C^aim 17. 19. A pharmaceutical compoεition compriεing the polypeptide of Claim 14 and a pharmaceutically acceptable carrier. 20. A method for determining an antagonist/inhibitor effective for preventing the action of hTopl-α comprising: combining DNA, hTopl-α and a potential antagonist/inhibitor; incubating the combination under effective conditions to allow for hTopl-α to act on the DNA; and determining if the antagonist/inhibitor effectively blocks the action of hTopl-α.

Inventors: ADAMS MARK D, FLEISCHMANN ROBERT D, WEI YING-FEI, ADAMS, MARK, D., FLEISCHMANN, ROBERT, D., WEI, YING-FEI
Applicants: ADAMS MARK D, FLEISCHMANN ROBERT D, HUMAN GENOME SCIENCES INC, WEI YING FEI, ADAMS, MARK, D., FLEISCHMANN, ROBERT, D., HUMAN GENOME SCIENCES, INC., WEI, YING-FEI
Application year: 1994
Main classification: C12N9
IPC codes: A61K31, A61K38, A61K39, A61K45, A61K48, A61P31, A61P35, A61P37, C07H21, C07K16, C12N5, C12N9, C12N15, C12P21, C12R1